Week 6 – Getting Down to Business

Week six of my internship was an exciting if rater uneventful week.  Monday was not too busy.  I transformed the Δ1990 DNA fragment into the E. coli competent cells that we made last week.  These then got plated onto an LB/agar plate containing Kanamycin.  We also sent in to be sequenced ten DNA fragment samples for the ΔtnbA gene, and I re-streaked ten new ΔtnbA colonies to use in case the sequencing results come back with mutations in all of the fragments.  Because we did not have a lot of work, I had some time to myself to work and read journal articles.

On Tuesday we received the second set of sequencing results for the ΔTERTU_2288 fragment had arrived yesterday, so I took a look at those.  One of the fragments showed no mutations from either direction.  Yay!  After I looked at these, I took the Δ1990 plate out, selected 25 colonies, and separated them out on a new agar plate.  This plate went into the 37°C incubator to grow until tomorrow morning.  When this was done, Hiroaki showed me how to start a colony PCR reaction on one of the DNA fragments he has been working on.  This step is used to determine which of the E. coli colonies actually contain the DNA fragment that we are interested in.  Once it was started I also started a digestion reaction on the ΔTERTU_2288 fragment to remove the fragment from the plasmid.    In the middle of the afternoon, I took the digestion reaction out of its water bath incubation and removed the enzyme using a PCR purification kit.  There was not enough time to start a second digestion reaction, so I put the fragment in the cooler until tomorrow morning. 

Wednesday was probably the busiest day of my entire internship so far.  At this point I have two separate fragments (Δ1990 and Δ2288) which do not contain mutations and which I am working with consistently.  On Wednesday, I had quite a bit to do for both fragments. 

For the Δ2288 fragment, I started the day with the second enzyme digestion to completely remove it from the plasmid.  Later in the day, I ran an agarose gel electrophoresis to separate the fragment from the plasmid and any remaining enzyme.  Then I extracted the ΔTERTU_2288 fragment from the gel and started the reaction to ligate the fragment into the suicide plasmid.  I put the reaction in the 4°C cooler to proceed overnight. 

For the Δ1990 fragment, I ran a colony PCR to determine which of the colonies contained the fragment.  I used the first ten colonies from the colony separation plate that I made yesterday morning.  All but two of the colonies showed positive results for presence of the fragment, so I picked three positive colonies and inoculated them in LB/Kanamycin broth.  I also re-streaked each colony from the plate made yesterday onto an ampicillin plate.  This step checks to make sure that none of the colonies contain the initial T-vector plasmid.    

Finally, before I left, I took a look at the sequencing results for the ΔtnbA fragment which had come in this afternoon.  Unfortunately, all of the fragments again showed mutations.

Thursday I got the chance to watch the PhD defense presentation of one of the graduate students in my lab, Christina.  This was really interesting and gave me a chance to see a portion of what it the process is like.  The presentation was a lot longer than I had expected, but I really enjoyed it, especially because my limited experience so far gave me a little bit of perspective into what the presentation was covering. 

Afterward, I extracted the DNA plasmid Δ1990 from two of the E. coli colony tubes that I had inoculated yesterday.  I also transformed these plasmids into a new E. coli strain (S17) which is capable of performing the necessary conjugation of our plasmid into the Teredinibacter turnerae bacteria.  I also transformed the ligated Δ2288 plasmid into the first strain of E. coli competent cells that we are using (DH5α, and plated both of these sets of E. coli.

Friday, similar to Monday was not too busy.  Most of the reactions that I need to do next involve several steps and require several consecutive days.  In the morning I made up a batch of LB/agar solution for making more Kanamycin plates and put that in the autoclave.  Then I washed the tubes from Wednesday’s inoculation and put them in a dishwashing tub.  I also separated out the DH5α colonies containing the Δ2288 fragment by streaking them onto an LB/Kanamycin agar plate from the 4°C room.  At noon all of the interns met for a seminar on work life balance.  After the seminar, I added Kanamycin to the LB agar solution and poured it into plates.